Richard M. Day

Collagen — Emerging collagen based therapies hit the patient

The choice of biomaterials available for regenerative medicine continues to grow rapidly, with new materials often claiming advantages over the short-comings of those already in existence. Going back to nature, collagen is one of the most abundant proteins in mammals and its role is essential to our way of life. It can therefore be obtained from many sources including porcine, bovine, equine or human and offer a great promise as a biomimetic scaffold for regenerative medicine. Using naturally derived collagen, extracellular matrices (ECMs), as surgical materials have become established practice for a number of years. For clinical use the goal has been to preserve as much of the composition and structure of the ECM as possible without adverse effects to the recipient. This review will therefore cover in-depth both naturally and synthetically produced collagen matrices. Furthermore the production of more sophisticated three dimensional collagen scaffolds that provide cues at nano-, micro- and meso-scale for molecules, cells, proteins and bulk fluids by inducing fibrils alignments, embossing and layered configuration through the application of plastic compression technology will be discussed in details. This review will also shed light on both naturally and synthetically derived collagen products that have been available in the market for several purposes including neural repair, as cosmetic for the treatment of dermatologic defects, haemostatic agents, mucosal wound dressing and guided bone regeneration membrane. There are other several potential applications of collagen still under investigations and they are also covered in this review.

Superparamagnetic iron oxide nanoparticle targeting of MSCs in vascular injury

Vascular occlusion can result in fatal myocardial infarction, stroke or loss of limb in peripheral arterial disease. Interventional balloon angioplasty is a common first line procedure for vascular disease treatment, but long term success is limited by restenosis and neointimal hyperplasia. Cellular therapies have been proposed to mitigate these issues; however efficacy is low, in part due to poor cell retention. We show that magnetic targeting of mesenchymal stem cells gives rise to a 6-fold increase in cell retention following balloon angioplasty in a rabbit model using a clinically applicable permanent magnet. Cells labelled with superparamagnetic iron oxide nanoparticles exhibit no negative effects on cell viability, differentiation or secretion patterns. The increase in stem cell retention leads to a reduction in restenosis three weeks after cell delivery.

Expression of syndecan-1 in inflammatory bowel disease and a possible mechanism of heparin therapy

Heparin apparently aids healing in ulcerativecolitis although its mechanism of action is unknown. Thepurpose of this study was to investigate the hypothesisthat heparin functions as a coreceptor molecule for basic fibroblast growth factor, a roleusually performed by heparan sulfate chains onsyndecan-1. A marked reduction of syndecan-1immunostaining was found in reparative epithelium frominflammatory bowel disease patients. Removal of heparansulfate on gastrointestinal epithelial cells in vitroreduced the proliferative response to basic fibroblastgrowth factor. The response to basic fibroblast growth factor was completely restored by the additionof heparin. Loss of syndecan-1 expression occurs in theregenerative mucosa in inflammatory bowel disease.Although this may facilitate tissue motility, its loss probably adversely affects the ability ofcells to bind basic fibroblast growth factor. Thepresent data show that heparin may substitute the lossof functional activity of syndecan-1 in the binding of basic fibroblast growth factor.

Cantharidin blisters: a technique for investigating leukocyte trafficking and cytokine production at sites of inflammation in humans

A skin blister technique is described which allows the investigation of acute inflammation in humans in vivo. Filter paper discs are placed on the skin, impregnated with cantharidin and covered with impermeable film held by adhesive tape. The assembly is easily applied, unobtrusive, stable and may be worn during normal activities. The blister formed at 24 h contains approximately 5x10(5)-5x10(6) cells, predominantly neutrophils and macrophages. Inflammatory cytokines and chemotactic factors are detectable in the blister fluid. The technique is useful for characterizing the acute inflammatory response in health and disease.

Impaired neutrophil chemotaxis in Crohn’s disease relates to reduced production of chemokines and can be augmented by granulocyte-colony stimulating factor

Defective neutrophil recruitment has been described as a primary pathogenic abnormality in Crohn’s disease. Cantharidin‐induced blisters provide a novel investigative tool to assess cellular influx and inflammatory mediator production during acute inflammation and allows the effects of therapy on these parameters to be measured.

Assessment of Polymer/Bioactive Glass-Composite Microporous Spheres for Tissue Regeneration Applications

Conformable scaffold materials capable of rapid vascularization and tissue infiltration would be of value in the therapy of inaccessible wounds. Microporous spheres of poly(D,L-lactide-co-glycolide) (PLGA) containing bioactive glass (BG) were prepared using a thermally induced phase separation (TIPS) technique, and the bioactivity, in vitro degradation, and tissue integration of the microporous spheres were assessed. Microporous spheres containing 10% (w/w) BG stimulated a significant increase in vascular endothelial growth factor secretion from myofibroblasts consistently over a 10-day period (p < 0.01) compared with the neat PLGA microporous spheres. The microporous spheres degraded steadily in vitro over a 16-week period, with the neat PLGA microporous spheres retaining 82% of their original weight and microporous spheres containing 10% (w/w) BG retaining 77%. Both types of microporous spheres followed a similar pattern of size reduction throughout the degradation study, resulting in a 23% and 20% reduction after 16 weeks for the neat PLGA microporous spheres and PLGA microporous spheres containing 10% (w/w) BG, respectively (p < 0.01). After in vivo implantation into a subcutaneous wound model, the TIPS microporous spheres became rapidly integrated (interspherically and intraspherically) with host tissue, including vascularization of voids inside the microporous sphere. The unique properties of TIPS microporous spheres make them ideally suited for regenerative medicine applications where tissue augmentation is required.

In vivo characterisation of a novel bioresorbable poly(lactide-co-glycolide) tubular foam scaffold for tissue engineering applications

Polylactide-co-glycolide (PLGA) foams of tubular shape were assessed for their use as soft- tissue engineering scaffolds in vitro and in vivo. Porous membranes were fabricated by a thermally induced phase separation process of PLGA solutions in dimethylcarbonate. The parameters investigated were the PLGA concentration and the casting volume of solution. Membranes produced from 5 wt/v % polymer solutions and a 6 ml casting volume of polymer solution were selected for fabricating tubes of 3 mm diameter, 20 mm length and a nominal wall thickness of 1.5 mm. Scanning electron microscopy revealed that the structure of the tubular foams consisted of radially oriented and highly interconnected pores with a large size distribution (50–300 μm). Selected tubes were implanted subcutaneously into adult male Lewis rats. Although the lumen of the tubes collapsed within one week of implantation, histological examination of the implanted scaffolds revealed that the foam tubes were well tolerated. Cellular infiltration into the foams, consisting mainly of fibrovascular tissue, was evident after two weeks and complete within eight weeks of implantation. The polymer was still evident in the scaffolds after eight weeks of implantation. The results from this study demonstrate that the PLGA tubular foams may be useful as soft-tissue engineering scaffolds with modification holding promise for the regeneration of tissues requiring a tubular shape scaffold such as intestine.

Regulation of epithelial syndecan-1 expression by inflammatory cytokines

Syndecan-1 is expressed on the basolateral surface of columnar epithelium and contributes to wound repair by facilitating increased growth factor binding. Inflammatory bowel disease (IBD) is associated with reduced syndecan-1 expression in areas of inflamed mucosa that is likely to impair mucosal healing. Reduced syndecan-1 expression in IBD may be related to the presence of increased inflammatory cytokines. To test this hypothesis, monolayers of HT29 and T84 colonic epithelial cells were stimulated with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta or IL-6. Stimulation of HT29 cells with TNF-alpha and IL-1beta resulted in reversible down-regulation of syndecan-1 at both protein and mRNA levels but little effect was observed with IL-6. Loss of syndecan-1 expression was caused by shedding of the ectodomain as revealed by increased levels of soluble syndecan-1 measured in the conditioned medium of stimulated cells. No increase in cytoplasmic staining accompanied the loss of cell surface syndecan-1 expression. TNF-alpha and IL-1beta are capable of down-regulating syndecan-1 expression and may account in part for the reduced expression of syndecan-1 seen in IBD.

Evaluation of decellularization protocols for production of tubular small intestine submucosa scaffolds for use in oesophageal tissue engineering

Small intestine submucosa (SIS) has emerged as one of a number of naturally derived extracellular matrix (ECM) biomaterials currently in clinical use. In addition to clinical applications, ECM materials form the basis for a variety of approaches within tissue engineering research. In our preliminary work it was found that SIS can be consistently and reliably made into tubular scaffolds which confer certain potential advantages. Given that decellularization protocols for SIS are applied to sheet-form SIS, it was hypothesized that a tubular-form SIS would behave differently to pre-existing protocols. In this work, tubular SIS was produced and decellularized by the conventional peracetic acid-agitation method, peracetic acid under perfusion along with two commonly used detergent-perfusion protocols. The aim of this was to produce a tubular SIS that was both adequately decellularized and possessing the mechanical properties which would make it a suitable scaffold for oesophageal tissue engineering, which was one of the goals of this work. Analysis was carried out via mechanical tensile testing, DNA quantification, scanning electron and light microscopy, and a metabolic assay, which was used to give an indication of the biocompatibility of each decellularization method. Both peracetic acid protocols were shown to be unsuitable methods with the agitation-protocol-produced SIS, which was poorly decellularized, and the perfusion protocol resulted in poor mechanical properties. Both detergent-based protocols produced well-decellularized SIS, with no adverse mechanical effects; however, one protocol emerged, SDS/Triton X-100, which proved superior in both respects. However, this SIS showed reduced metabolic activity, and this cytotoxic effect was attributed to residual reagents. Consequently, the use of SIS produced using the detergent SD as the decellularization agent was deemed to be the most suitable, although the elimination of the DNase enzyme would give further improvement.

Microporous collagen spheres produced via thermally induced phase separation for tissue regeneration

Collagen is an abundant protein found in the extracellular matrix of many tissues. Due to its biocompatibility, it is a potentially ideal biomaterial for many tissue engineering applications. However, harvested collagen often requires restructuring into a three-dimensional matrix to facilitate applications such as implantation into poorly accessible tissue cavities. The aim of the current study was to produce a conformable collagen-based scaffold material capable of supporting tissue regeneration for use in wound repair applications. Microporous collagen spheres were prepared using a thermally induced phase separation (TIPS) technique and their biocompatibility was assessed. The collagen spheres were successfully cross-linked with glutaraldehyde vapour, rendering them mechanically more stable. When cultured with myofibroblasts the collagen spheres stimulated a prolonged significant increase in secretion of the angiogenic growth factor, vascular endothelial growth factor (VEGF), compared with cells alone. Control polycaprolactone (PCL) spheres failed to stimulate a similar prolonged increase in VEGF secretion. An enhanced angiogenic effect was also seen in vivo using the chick embryo chorioallantoic membrane assay, where a significant increase in the number of blood vessels converging towards collagen spheres was observed compared with control PCL spheres. The results from this study indicate that microporous collagen spheres produced using TIPS are biologically active and could offer a novel conformable scaffold for tissue regeneration in poorly accessible wounds.